Project Summary BRAF inhibitors lack efficacy in BRAF mutant (BRAFm) CRC (response rate only 5%) in contrast to response rates of >50% in BRAFm melanoma. Key studies conducted as part of our prior SPORE project identified feedback networks present in CRC (but absent in melanoma) that lead to rapid reactivation of MAPK signaling following BRAF inhibition, as primary drivers of resistance. This critical discovery led to clinical trials of BRAFi-based therapeutic combinations designed to block MAPK reactivation, resulting in an increased response rate for BRAFm CRC patients from 5% to >30%. Despite these therapeutic advances, clinical benefit is not durable, with a median PFS of only 4-5 months. Here we will explore potential cooperativity between targeted MAPK inhibition (MAPKi) and immune checkpoint blockade (ICB) to convert less immune responsive tumors to more immunogenic tumors. BRAFm CRC represents a prime population for exploring potential cooperativity, as 20-30% of metastatic BRAFm CRCs harbor MSI, which confers responsiveness to ICB. Moreover, we have observed durable responses of >5 years in MSI BRAFm CRC patients receiving MAPKi alone. In MSS BRAFm CRC patients, we see marked induction of CD4+ and CD8+ T-cells with MAPKi alone in paired tumor biopsies, and our preclinical mouse models demonstrate a cooperative effect of MAPKi and PD-1 IC in MSS BRAFm CRC. We propose a comprehensive effort using innovative immune competent BRAFm CRC mouse models, cutting-edge molecular and immune analyses of paired pre- and on-treatment tumor biopsies, and novel clinical trials to explore combined MAPKi and ICB as a strategy to achieve durable benefit in BRAFm CRC patients. Aim 1 will define the effects of MAPKi alone and with PD-1 ICB on immunogenicity of BRAFm CRC and anti-tumor immunity using immunologic and transcriptional profiling approaches to analyze novel BRAFm CRC models and a unique collection of paired pre-treatment and on-treatment biopsies from BRAFm CRC patients given BRAF/EGFR/MEKi. Aim 2 will conduct clinical trials and correlative studies of novel immune and targeted combinations for BRAFm CRC, evaluating clinical efficacy of combined BRAF/MEK/PD-1 inhibition. We will collaborate with the Pathology Core for multiplexed immune analysis of tumor biopsies, and the Biostats Core for analysis of bulk and single cell RNAseq and whole-exome sequencing. These studies will provide key insights to guide design of future trials. Aim 3 will define mechanisms of response and resistance to combined MAPKi and ICB in BRAFm CRC mouse models, and test strategies to overcome resistance to MAPKi/anti-PD-1 using combined ICB and modulators of immunosuppressive mechanisms defined by our analyses in Aims 1 and 2. These studies will define the potential synergy between MAPKi and ICB in BRAFm CRC and mechanisms of response and resistance to establish a new therapeutic paradigm for this lethal CRC subtype